Panoply™ Human PKLR Knockdown Stable Cell Line

The conventional treatment for prostate cancer (PCa) involves androgen deprivation therapy (ADT) to suppress androgen receptor (AR) signaling pathway-driven tumor progression. ADT-induced PCa relapse may progress to an AR-negative phenotype with neuroendocrine (NE) histology, which is associated with metabolic disturbances and poor prognosis. However, the metabolic pathways regulating NE differentiation (NED) in PCa remain unclear. Here, researchers reveal the regulatory mechanism of ADT-induced NED-related metabolic dysfunction: overexpression of pyruvate kinase L/R (PKLR) promotes NED and tumor invasiveness by upregulating reactive oxygen species regulator 1 (ROMO1) to mediate oxidative stress. ADT mediates nuclear translocation of PKLR, which binds to the MYCN/MAX complex, upregulating ROMO1 and NE-related genes, leading to mitochondrial functional alterations and NED in PCa. Targeting the nuclear PKLR/MYCN pathway with bromine domain and terminal outer motif (BET) inhibitors holds promise for reducing PKLR/MYCN-driven neuroendocrine differentiation (NED). Abundant ROMO1 in serum samples may provide prognostic information for patients receiving androgen deprivation therapy (ADT). These findings suggest that ADT resistance leads to upregulation of the PKLR/MYCN/ROMO1 signaling pathway, which may drive metabolic reprogramming and NED in prostate cancer (PCa).

To determine the correlation between PKLR and NEPC status, researchers validated PKLR expression levels in a range of PCa cell lines. The results showed that elevated PKLR expression was associated with NE markers and was enriched in MDVR-resistant C4-2-MDVR cells, AR-negative PC3 cells, and NEPC-like LASCPC01 cells, while PKLR expression was lower in AR-positive LNCaP cells and C4-2 cells (Figure 1A). In PKLR-knockdown PC3 cells, PKLR inhibition was associated with decreased NE markers, increased autophagy/mitochondrial autophagy, and apoptosis markers, but no change in AR levels (Figure 1B). To determine the role of PKLR in mediating mitochondrial function in PCa cells, researchers validated the mtDNA content in PKLR-knockdown PC3 cells, finding that decreased mtDNA content was associated with decreased ATP levels compared to control cells (Figures 1C and D). Next, they analyzed the oxygen consumption rate (OCR) of PKLR-knockdown PC3 cells using a Seahorse XF24 analyzer to determine the role of PKLR in mitochondrial respiration. The results showed that the OCR value of PKLR knockdown cells was significantly reduced compared with the control group (Figure 1E). Subsequently, they examined the effect of PKLR knockdown on PC3 cell proliferation, finding that both the proliferation capacity and the number of 3D spheroids were reduced in PKLR knockdown cells (Figure 1F). The migration and invasion abilities of PKLR knockdown cells were also reduced in Matrigel (Figure 1G). Furthermore, flow cytometry analysis showed that PKLR knockdown cells produced reduced reactive oxygen species (ROS) and their cell cycle arrested in the G1 phase (Figure 1H).

Figure 1. PKLR upregulates mitochondrial function and increases aggressiveness of prostate cancer (PCa) cells. (Chen W Y, et al., 2023)